Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases

Oz, Iris; Avidan, Orna; Hizi, Amnon

doi:10.1042/bj3610557

Cited by 33 publications

(44 citation statements)

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“…LEDGF 402-411 inhibited the enzymatic activity of IN more potently than LEDGF 361-370 did, although its affinity to IN was 3-fold weaker. This observation is because binding affinity is not the only factor that is responsible for the inhibitory activity of a molecule (K D and K i are different in many cases), as was shown for the RT inhibitors (36,37). The peptides inhibit integration in cells, do not lower the amounts of reverse transcripts, but do reduce the number of proviral copies and HIV-1 Tat-mediated transcription, showing that the peptides do not affect the early, but do halt the late, events of HIV-1 replication.…”

Section: Discussionmentioning

confidence: 96%

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Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

Hayouka

Rosenbluh²,

Levin³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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172

202

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Proteins are involved in various equilibria that play a major role in their activity or regulation. The design of molecules that shift such equilibria is of great therapeutic potential. This fact was demonstrated in the cases of allosteric inhibitors, which shift the equilibrium between active and inactive (R and T) states, and chemical chaperones, which shift folding equilibrium of proteins. Here, we expand these concepts and propose the shifting of oligomerization equilibrium of proteins as a general methodology for drug design. We present a strategy for inhibiting proteins by ''shiftides'': ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein toward it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein by using peptides derived from its cellularbinding protein, LEDGF/p75, which specifically inhibit IN activity by a noncompetitive mechanism. The peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer toward the inactive tetramer, which is unable to catalyze the first integration step of 3 end processing. The LEDGF/ p75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells because of the lack of integration. These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a previously uncharacterized mechanism, which bears advantages over the conventional interface dimerization inhibitors.allostery ͉ protein equilibrium ͉ shiftides ͉ peptides ͉ drug design

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Section: Discussionmentioning

confidence: 96%

“…The 3Ј end processing and the strandtransfer activities of IN were performed as previously described (36,37).…”

Section: Methodsmentioning

confidence: 99%

Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

Hayouka

Rosenbluh²,

Levin³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

172

202

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“…19,20 In contrast, Hehl et al have shown that with different assay and buffer conditions, RT at different molar ratios enhances strand transfer. 21 As a result, we were interested in how RT behaved in our HITS™ assay.…”

Section: Reverse Transcriptase Inhibits Integrationmentioning

confidence: 99%

“…This result agrees with the previously published data. 19,20 Since the activity of IN might be different at physiological temperature, we checked the effect of preincubation of the reaction at 37°C, using the microplate assay. In contrast to what we observed with preincubation of IN with DNA at 4°C, RT did not block integration when IN was preincubated with DNA at 37°C for 5 min.…”

Section: Reverse Transcriptase Inhibits Integrationmentioning

confidence: 99%

Development and Application of a High-Throughput Screening Assay for HIV-1 Integrase Enzyme Activities

2005

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Integrase (IN) mediates the covalent insertion of the retroviral genome into its host chromosomal DNA. This enzymatic activity can be reconstituted in vitro with short DNA oligonucleotides, which mimic a single viral DNA end, and purified IN. Herein we report a highly efficient and sensitive high-throughput screen, HIV Integrase Target SRI Assay (HITS™), for HIV-1 IN activity using 5′ biotin-labeled DNA (5′ BIO donor) and 3′ digoxygenin-labeled DNA (3′ DIG target). Following 3′ processing of the 5′ BIO donor, strand transfer proceeds with integration of the 5′ BIO donor into the 3′ DIG target. Products were captured on a streptavidin-coated microplate and the amount of DIG retained in the well was measured. The end point values, measured as absorbance, ranged from 0.9 to 1.5 for IN-mediated reactions as compared with background readings of 0.05 to 0.12. The Z factor for the assay ranged from 0.7 to 0.85. The assay was used to screen drugs in a high-throughput format, and furthermore, we adapted the assay to study mechanistic questions regarding the integration process. For example, using variations of the assay format, we showed high preference of E strand of the long terminal repeat (LTR) viral DNA as a target strand compared with its complementary A strand. The E strand is the strand processed by IN. Furthermore, we explored the reported inhibitory effect of reverse transcriptase on integration. (Journal of Biomolecular Screening 2005:606-614)

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“…For example, it has been reported that NCp7 might influence the coupled joining by promoting DNA distortion (16) and that reverse transcriptase might play a role in blocking the auto-integration process (17). Host proteins, upon association with IN, have also been reported to improve in vitro viral DNA integration.…”

Section: Hiv-1 1 Integrase (In) Is An Essential Component Required Fomentioning

confidence: 99%

Reversion of the Lethal Phenotype of an HIV-1 Integrase Mutant Virus by Overexpression of the Same Integrase Mutant Protein

Priet

Navarro²,

Quérat³

et al. 2003

Journal of Biological Chemistry

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Inhibition of the integrases of human immunodeficiency viruses type 1 and type 2 by reverse transcriptases

Cited by 33 publications

References 0 publications

Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

Development and Application of a High-Throughput Screening Assay for HIV-1 Integrase Enzyme Activities

Reversion of the Lethal Phenotype of an HIV-1 Integrase Mutant Virus by Overexpression of the Same Integrase Mutant Protein

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